ARTICLE https://doi.org/10.1038/s41467-021-21120-8 OPEN Mechanism of the small ATP-independent chaperone Spy is substrate specific Rishav Mitra1,2, Varun V. Gadkari3, Ben A. Meinen1,2, Carlo P. M. van Mierlo 4, Brandon T. Ruotolo 3 & ✉ James C. A. Bardwell1,2 ATP-independent chaperones are usually considered to be holdases that rapidly bind to non- native states of substrate proteins and prevent their aggregation. These chaperones are 1234567890():,; thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action. 1 Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA. 2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. 3 Department of Chemistry, University of Michigan, Ann Arbor, MI, USA. 4 Laboratory of Biochemistry, Wageningen ✉ University, Wageningen, The Netherlands. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:851 | https://doi.org/10.1038/s41467-021-21120-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21120-8 opologically complex proteins often populate misfolded bound to the chaperone10. The substrates studied in detail so far, intermediates that act as kinetic traps1.Suchinter- namely Im7 and SH3, are small proteins (10 and 7 kDa, respec- T α β mediates often expose hydrophobic surfaces that make tively) with very simple 3D structures (just -helices or just - them prone to aggregation. ATP-dependent molecular cha- strands, respectively). We wondered if the folding-while-bound perones like GroEL-GroES rescue trapped intermediates and mechanism of Spy applies to more complex substrates that include facilitate substrate folding2. In contrast, ATP-independent both α-helices and β-strands and have a more complex folding chaperones generally bind very tightly to non-native sub- pathway than the substrate proteins tested so far. To investigate this, strates and in doing so prevent protein aggregation but are not we chose the apoflavodoxin AnFld as our substrate, as its native thought to directly facilitate substrate refolding3.Thesimple state has an α–β parallel topology and as apoflavodoxin exhibits a designations of “foldase” or “holdase” may underemphasize the more complex folding pathway than either Im7 or SH3 in that it microscopic structural heterogeneity of chaperone–substrate folds via a three-state triangular mechanism with an essentially off- complexes4. Substrates bound to ATP-independent chaperones pathway intermediate (Fig. 1a)11. In this mechanism, most of the such as trigger factor, SecB, and the sHsps can adopt a wide molecules in the intermediate conformation need to unfold prior to range of conformations ranging from near-native to unfolded refolding into the native conformation. A small fraction of mole- states5–7. We have shown that the ATP-independent chaperone cules can directly fold from the intermediate to the native state. Spy not only binds protein folding intermediates of substrate First, we verified that the previously established triangular folding proteins such as Im7 and SH3 but also allows for the folding of mechanism for flavodoxin folding is functional under our buffer substrate proteins while they remain chaperone bound8,9.Spy conditions (40 mM HEPES-KOH pH 7.5, 100 mM NaCl), which we loosely binds to different folding states of these model substrate considered to be more physiological than the previously used buffer proteins.Althoughthefoldingrateconstantforthesetwo (50 mM MOPS pH 7.0) (Fig. S1a–d)8,11. We studied the refolding simple substrates decreases with increasing concentrations of of AnFld in the presence of Spy by monitoring the tryptophan Spy, it does not go to zero at saturating Spy concentrations fluorescence in a stopped-flow fluorimeter upon diluting urea- where essentially all the substrate molecules are chaperone denatured AnFld into refolding buffer containing increasing bound10, evidence that folding of the substrate occurs while it concentrations of Spy, as described previously8. We observe two chaperone-bound. In this mechanism, substrate release is not a kinetic phases of AnFld folding; a major kinetic phase, where the prerequisite for substrate folding. We have found that the amplitude is proportional to the fraction of denatured molecules folding-while-bound mechanism is dependent on relatively that directly fold to the native state (N), and a minor phase whose weak chaperone–substrate interactions9.VariantsofSpythat amplitude is proportional to the fraction of molecules that bind SH3 with stronger affinity than does wild-type (WT) Spy transiently populate the off-pathway intermediate and therefore significantly slow the folding of bound substrate9.Thesub- need to unfold before productive refolding11. In the presence of Spy, strates of Spy tested so far have been small, topologically sim- the observed rate constants (kobs) of both phases decrease ple, all β-sheet or all α-helical model proteins. We wanted to substantially until at the highest Spy concentration (~9 μM), a −1 test if the folding-while-bound model is generalizable to larger single folding phase remains that has a small kobs value (0.1 s ) substrates with complex topologies such as those found in the (Fig. 1b, c). This indicates that Spy significantly slows the folding vast majority of proteins. Here, we chose apoflavodoxin from rate of AnFld, like for Im7 and SH3. However, both Im7 and SH3 Anabaena PCC 7119 (AnFld) and Azotobacter vinelandii can fold to the native state, albeit slowly, even at saturating Spy (AzoFld), both well-studied folding models11,12 that populate concentrations8,9. In contrast, binding to Spy prevents folding of kinetically trapped off-pathway intermediates13,14.Theirα/β AnFld almost completely, as evidenced by the dramatic decrease in topology is both ancient and frequently found in common folds the folding amplitude upon increasing Spy concentration (Fig. 1b). such as TIM barrels, Rossman folds, FAD/NAD(P)-binding At Spy concentrations higher than ~9 μM, the amplitude of the domains, and P-loop containing hydrolases. The goal of this fluorescence change was extremely small, indicating that most of study was to test whether the folding-while-bound mechanism the denatured AnFld molecules are kinetically trapped in a folding- adequately describes the effect of Spy on the folding of proteins incompetent bound state. Both the unfolded (U) and intermediate with complex topologies using the flavodoxin-like fold as an (I) states of AnFld are less fluorescent than the native state11.Since example. Our results show that Spy’s mechanism is surprisingly AnFld in the presence of Spy is substantially less fluorescent than it substrate specific.Inthecaseofapoflavodoxin, Spy traps is in the native state, we reason that Spy kinetically traps AnFld in a denatured polypeptides in a non-native state, thereby inhibiting non-native state that is partially or completely unfolded (we term flavodoxin folding. This dual functionality of Spy as both a this state U*). It appears that Spy binds very rapidly to AnFld and folding-while-bound chaperone and a holdase rests on oppos- prevents folding since the fluorescence decreases within the ~25 ms ing thermodynamic requirements. The folding-while-bound dead time of the stopped-flow instrument. The interaction of Spy * paradigm requires that Spy bind the various folding states of and the U state is tight with a dissociation constant (KD)of0.35 the substrate weakly. In contrast, a holdase binds non-native μM(Fig.1d). The affinity of Spy for AnFldU* in folding states tightly such that folding transitions are not feasibly experiments is ~13-fold tighter than observed for Spy binding to = μ thermodynamically. We show that the mechanism of action of the intermediate and unfolded states of Im7 (KD 4.7 M for both) the chaperone Spy is dictated by the difference in binding and ~8-fold tighter than that between Spy and unfolded SH3 fi = μ 8,9 af nities for partially unfolded states of different substrates to (KD 2.9 M), both of which fold while bound to Spy .The Spy. This work highlights the need to shift from monolithic kinetic trapping of AnFld in the Spy-bound U* state due to these enzyme-inspired views of molecular chaperones toward more high-affinity interactions apparently necessitates release from Spy complex models that incorporate the possibility of multiple before productive folding can occur. substrate-specific mechanisms. Rapid binding of Spy is coupled to partial unfolding of Results apoflavodoxin. Having shown that Spy inhibits the folding of Spy inhibits the folding of apoflavodoxin by kinetically trap- AnFld, we wondered if Spy interacts with AnFld’s native state. ping it in a non-native state. Spy belongs to an emerging class of Holdase chaperones are known to preferentially bind to non- chaperones that allow protein folding while the substrate remains native unfolded or misfolded proteins15. Folding-while-bound 2 NATURE COMMUNICATIONS | (2021) 12:851 | https://doi.org/10.1038/s41467-021-21120-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21120-8 ARTICLE Fig. 1 Kinetics of apoflavodoxin folding in the presence of Spy. a Folding pathway of apoflavodoxin from Anabaena PCC 7119 (AnFld) and Azotobacter 11, 12 vinelandii (AzoFld) . N, U, and I are the native, unfolded, and intermediate states, respectively.